“两步转化法”高效制备携带自杀基因的重组腺病毒载体

Two-step transformation for highly efficient construction of a replication-defective recombinant adenoviral vector

目的采用“两步转化法”高效制备含胞嘧啶脱氨酶(CD)自杀基因的腺病毒重组载体。方法将质粒pAdEasy-1线性化后转化于BJ5183菌,制备AdEasy-1细菌;自载体pBS-CD中切出CD基因,亚克隆至穿梭质粒pAdtrackCMV上,构建的pAdtrackCMV-CD线性化后转化AdEasy-1细菌,抽提同源重组腺病毒质粒,PacⅠ酶切后转染293细胞,在293细胞中包装与扩增,利用GFP报告基因进行滴度测定。同时按“一步转化法”进行同样的重组腺病毒制备,比较二者的优劣性。结果“两步转化法”终产物17个克隆中13个正确,正确率为76.5%(13/17);“一步转化法”重组产物17个克隆中有2个正确,正确率为11.8%(2/17),两者具有显著性差异(P=0.000 17)。结论“两步转化法”细菌内同源重组是一种更高效、更方便的重组腺病毒制备方法。

Objective To efficiently construct a replication-defective recombinant adenoviral vector using a two-step transforma- tion procedure. Methods Plasmid pAdEasy-1 was linearized and transformed into E.coli BJ5183 to construct BJ5183pAdEasy-1 cells. Cytosine deaminase (CD) gene was obtained from plasmid pBS-CD, and subcloned into the shuttle plasmid to form transfer plamid of pAdtrackCMV-CD, which was then linearized and transformed into BJ5183pAdEasy-1 cells. The recom- binant adenovirus plasmid DNA was extracted from the transformed bacteria and digested with PacⅠ after identification, followed by transfection into 293 packaging cells. PCR was used to detect target gene, and the titer and the infection rate of the recombinant Ad were measured with the aid of green fluorescent protein (GFP) reporter gene. The same recombinant AdCMV-CD was constructed in a one-step transformation method for comparison. Results The homologous recombination of the two-step transformation method resulted in a success rate of 76.5% (13/17), while the success rate of the one-step method was only 11.8% (2/17), showing significant difference between the two methods (P=0.000 17 by Fisher's exact test). Conclusion The two-step transformation procedure is more efficient and convenient than one-step method.